Seasonal influenza (flu) infections kill hundreds of thousands of people throughout the world each year and morbidity and mortality rates increase even further when a new pandemic virus emerges. The annual flu vaccine provides an important line of defense against influenza infections but there are many documented examples of poor vaccine efficacy, even in healthy individuals. Thus, there is an unmet public health need to improve influenza vaccination. However, before we can improve the vaccine, we must first better understand the immune cells that respond to the vaccine. One cell type of particular importance is the memory B cell, which is rapidly recalled following infection and vaccination and can contribute to anti-viral immunity by differentiating into short-lived antibody secreting cells (ASCs) that produce antibodies (Ab) that can either neutralize the virus or facilitate its rapid clearance. Memory B cells also seed the vaccine-driven humoral immune response and can give rise to new cohorts of long-lived ASCs and memory B cells that will protect against future exposures to the circulating flu virus. Despite the fact that memory B cells are the direct targets of vaccination in adults, we know remarkably little about these cells. This substantial knowledge gap is due, at least in part, to an inability to isolate and characterize these cells. Studies of memory B cells in humans have been even more difficult as we can typically only look at the cells in the blood and many immune cells, including the memory B cells, reside in tissues that are usually inaccessible in humans. We developed new tools to track flu-specific memory B cells in both mice and humans and have now identified and characterized distinct populations in the tissues of mice that we believe are likely to contribute to long-lasting vaccine induced immune responses. The central hypothesis of this application is that a flu-specific memory B cell subset that expresses the transcription factor T-bet is maintained long-term in tissues like the lung and is preferentially recalled by vaccination. The specific goals for this proposal are to identify the vaccine-elicited signals required to establish lung-resident T-bet expressing memory B cells, to determine whether vaccination and infection induce distinct populations of memory B cell with differing functions and life- spans, to identify which memory B cell subsets are preferentially recalled following antigen exposure, and to evaluate whether the repertoire of the T-bet expressing memory B cells found in human tissues are enriched for reactivity to older ?historic? flu antigens that the individual likely encountered early in life. The long term goal of this proposal is to fill our gap in our knowledge by developing the tools and animal model systems that will allow us to systematically and comprehensively study the heterogeneous populations of flu-specific memory B cells that persist in lymphoid and non-lymphoid tissues and are the direct targets of vaccination in adults. We believe that this contribution is significant as we expect to provide new insight into the complex and heterogeneous memory B compartment that is critical for vaccine-induced responses to flu.